引物延伸预扩增在β-地中海贫血产前基因诊断中的应用研究

目的探讨孕妇外周血中单个胎儿有核红细胞(NRBC)经引物延伸预扩增(PEP)后在β-地中海贫血产前基因诊断中应用的可行性。方法以2004年7月至2005年6月在广西医科大学第一附属医院行产前诊断,夫妇均为轻型β-地中海贫血,孕龄9～34周的28例孕妇为研究对象。显微操作获取母血中NRBC,PEP对单个NRBC进行全基因组扩增,短串联重复序列(STR)鉴定所取细胞的来源。证实为胎儿细胞的PEP产物作为模板进行β-珠蛋白基因扩增,反向点杂交确定胎儿β-珠蛋白基因型。结果每例发现NRBC4～13个,约43.6%的NRBC来源于胎儿。单个NRBC经PEP后STR及β-珠蛋白基因扩增成功率分别为100.0%和90.8%。经反向点杂交可鉴定胎儿β-珠蛋白基因点突变类型,与传统的产前诊断结果相比较,符合率为85.7%,误诊率14.3%。结论母血中单个胎儿NRBC经PEP后可以进行产前基因诊断,不仅可以应用于β-地中海贫血,也可应用于其它遗传病,是一条可供尝试的无创性产前诊断途径。     

应用磁珠细胞分离仪从母血中分离胎儿细胞的研究

目的 研究利用免疫磁珠细胞分离仪分离孕妇血中胎儿细胞的效率。方法 孕妇血采自孕龄为21－40周，初孕的孕男性单胎正常孕妇16例。脐血来自胎龄38－40周的男性新生儿10例。标本根据CD71抗体的效价标记磁珠后效在磁场中进行磁珠细胞分离。分离后的脐血标本在流式细胞仪上检测分离效率，孕妇血标本经套式PCR方法鉴定男性SRY基因片段，根据CD71－FITC抗体效价标记脐血中的单个核细胞。同时利用流式细胞仪检测和分离脐血中的胎儿细胞。结果 利用磙珠细胞分离仪分离孕妇血中的胎儿有核红细胞，经套式PCR技术寻找男性胎儿SRY基因片段，经第一次扩增有效率87.5%，第二次扩增有效率100.0%。脐血中胎儿有核红细胞在分离前占53.2%－74.9%；分离后，流式细胞仪富集为99.2%－99.8％，免疫磁珠细胞分离仪富集为99.2%－99.8%，免疫磁珠细胞分离仪富集为97.3%-99.4%。结论 免疫磁珠细胞分离仪可富集到98.3%的胎儿有核红细胞，经套式PCR技术在分离后的孕妇血中可确定100％的男性胎儿SRY基因片段，项技术简单，价廉，高效更适用于临床。     

孕妇外周血中胎儿有核红细胞的富集纯化及在产前诊断中的应用

孕妇外周血中的胎儿细胞主要有三种:滋养叶细胞、有核红细胞和淋巴细胞。最适于富集纯化用于产前诊断的是胎儿有核红细胞。密度梯度离心富集结合胎儿有核红细胞特异性抗原标记CD71,γ珠蛋白阳性选择和白细胞抗原CD45阴性选择纯化是目前从孕妇外周血中分离胎儿有核红细胞的理想方法。已用于产前诊断胎儿性别和一些疾病。     

STR-PCR在产前诊断胎儿唐氏综合征中的应用

目的:研究通过多重荧光定量PCR诊断胎儿染色体非整倍体用于临床快速产前诊断的可行性。方法:从孕中期羊水中提取胎儿DNA,通过多重荧光定量PCR使用STR对13、18、21号染色体进行非整倍体筛查,筛查结果异常者再进行快速诊断。用PCR诊断的羊水标本同时使用"金标准"染色体核型分析法做对比。结果:34例羊水标本中2例标本由于母血污染严重未行PCR检测,1例标本经PCR及核型分析均失败,29例标本经PCR和核型分析诊断为正常染色体,2例标本经PCR和核型分析诊断为21-三体。结论:通过STR-PCR法使用多重荧光酶联聚合反应探针产前诊断胎儿唐氏综合征是临床快速产前诊断的有效方法之一。     

母血中胎儿有核红细胞数量与胎儿异常的关系

目的 :探讨母血中胎儿有核红细胞数量与胎儿异常的关系 ,为将它用于无创性产前诊断提供实验依据。方法 :对孕 6～ 40周的 86例妇女 (包括胎儿正常组 68例、胎儿异常组 1 8例 )的外周血进行单密度梯度离心、瑞氏染色和细胞计数 ,分析母血中胎儿有核红细胞数量与孕周、胎儿异常及胎儿性别的关系。结果 :胎儿正常组 ,母血中胎儿有核红细胞平均数量为 1 4.2 3±1 2 .0 1 /ml,与妊娠周数呈直线相关 (R >R0 .0 5)。胎儿异常组中 ,母血中胎儿有核红细胞平均数量较正常胎儿组显著增加 ,约为 3 8.73± 2 4.97/ml,且与妊娠周数无明显直线相关性。母体外周血中胎儿有核红细胞数量与胎儿性别无统计学相关性。结论 :胎儿发生异常时母血中胎儿有核红细胞数量较正常妊娠时增加。母血中胎儿有核红细胞数量在胎儿正常时随孕周而增加 ,且不受胎儿性别的影响。母血中胎儿有核红细胞计数可以辅助用于产前筛查胎儿异常的发生。     

运用孕妇外周血中胎儿细胞产前诊断弓形虫感染的方法分析

弓形虫病(toxoplasmosis,TOX)是TORCH病毒感染中唯一的人畜共患病,在人群中具有普遍易感性,育龄妇女的弓形虫感染可能引起严重的胎儿损害,导致流产、死胎、胎儿畸形、胎儿生长受限及围生儿死亡等。目前,临床主要运用孕妇血清,羊水和脐血等诊断胎儿的感染情况,均具有不确定性和创伤性。本研究应用引物延伸预扩增和PCR方法运用母血中单个胎儿有核红细胞(fetal nucleated redblood cell,FNRBC)诊断出胎儿弓形虫感染一例,是一种无创性产前诊断弓形虫感染的新方法。1资料与方法1.1研究对象2005年10月至2006年6月在我院妇产科门诊优生实验…     

利用PRINS技术进行无创性产前诊断胎儿非整倍体

目的利用引物原位合成术(primedin situ labelling,PRINS)进行产前诊断胎儿非整倍体,探索无创性产前诊断的可靠便捷的新方法。方法采用流式细胞术从120例孕妇外周血中分离出胎儿有核红细胞,应用PRINS技术分别检测胎儿有核红细胞内的X、Y及21号染色体。结果120例标本中每例都可以检测出X染色体,敏感性和特异性均为100%;检测出Y染色体69例,敏感性92%(69/75),特异性为100%(69/69),检测出Klinefelter(XXY)综合征1例,唐氏综合征2例。结论应用PRINS技术对孕妇外周血中的胎儿细胞进行无创性产前诊断胎儿非整倍体是一种快速、敏感性高、特异性强的新方法,具有应用于临床的前景与价值。     

富集孕妇外周血中胎儿细胞行胎儿性别诊断

目的探讨对孕妇外周血中胎儿细胞进行非侵入性产前诊断的可行性。方法对６４例孕８～４０周孕妇外周血进行密度梯度离心法,富集胎儿细胞,并经显微镜下观察计数各种细胞所占百分比。同时,对所富集细胞经提取ＤＮＡ进行人Ｙ染色体特异ＤＮＡ扩增,判定胎儿性别并与新生儿性别进行对照。结果６４例孕妇外周血中,２５例观察到有核红细胞,占３９．０６％。６４例孕妇分娩３３例男性婴儿,其中２８例扩增出Ｙ特异带;另外３１例分娩女性婴儿,除１例出现Ｙ特异带,其余未见特异带。诊断的灵敏度为８４．８５％,特异度为９６．７７％,总符合率为９０．６３％。结论密度梯度离心法可从孕妇血中富集胎儿细胞,且所获得细胞已基本满足体外扩增Ｙ染色体基因所需要的模板量。     

微浓缩母体血获得胎儿细胞产前诊断13三体

检测早孕期母体血液中胎儿有核红细胞染色体已成为一种非损伤性的诊断方法,目前已成功应用于21三体、18三体以及性染色体非整倍体的产前诊断。作者首次报道通过FISH分析母血中胎儿有核红细胞(NRBC)产前诊断1例13三体。 病例39岁,健康高龄产妇,孕11周时接受宫颈绒毛膜绒毛取样(CVS)25mg,而且在CVS前后20分钟各抽母血15ml。2份血液标本按已报道的方法用菲科尔(ficoll)三倍密度梯度法浓缩NRBC,离     

孕妇外周血中胎儿有核红细胞的富集纯化及在产前诊断中…

孕妇外周血中的胎儿细胞主要有三种：滋养叶细胞、有核红细胞和淋巴细胞，最适于富集纯化用于产前诊断的是胎儿有核红细胞，密度梯度离心富集结合胎儿有核红细胞特异性抗原标记ＣＤ７１，γ株蛋白阳性选择和白细胞抗原ＣＤ４５阴性选择纯性是目送目前从孕妇外周血中分离胎儿有核红细胞的理想方法。已用于产前诊断胎儿性别和一些疾病。     

Diagnosis of human cytomegalovirus intrauterine infection using fetal cells from maternal blood.

OBJECTIVE: The sensitivity and specificity for the noninvasive prenatal diagnosis of human cytomegalovirus intrauterine infection were estimated by using isolating single fetal cells from maternal peripheral blood. METHODS: Micromanipulation techniques were employed to isolate single fetal nucleated erythroblasts from 273 maternal blood samples. SRY gene and HCMV-DNA in single fetal cells were detected by multiple primed in situ labeling (PRINS) from 76 HCMV-DNA positive samples of maternal peripheral blood. 273 samples of maternal peripheral blood were tested for SRY gene and HCMV-DNA in single fetal cells by primed extension preamplification (PEP) and polymerase chain reaction (PCR). RESULTS: The detection rate of fetal cells from maternal blood was 100% with micromanipulation techniques. The sensitivity of PRINS for SRY gene detection was 97.56% and its specificity was 100%. The sensitivity and specificity of PEP and PCR for SRY gene detection were 97.39% and 99.17%, respectively. The sensitivity of PRINS for HCMV-DNA detection was 92.68% and the specificity was 100%. The sensitivity and specificity of PEP and PCR for HCMV-DNA detection were 95.12%and 100%, respectively. CONCLUSION: The technique for noninvasive prenatal detection of intrauterine infection of HCMV using single fetal cells from maternal peripheral blood by using PRINS and PEP and PCR is more reliable than the CMV-DNA detection in peripheral maternal blood, amniocentesis or percutaneous umbilical blood sampling.     

Fetal origin of single nucleated erythroblasts and free DNA in the peripheral blood of pregnant women.

OBJECTIVES: To investigate the feasibility of using single fetal nucleated erythroblasts (FNRBCs) and free DNA in maternal blood for non-invasive prenatal diagnosis. METHODS: Single FNRBCs were isolated from 51 of 116 samples of maternal blood analyzed by micromanipulation after density gradient centrifugation. Furthermore, the nested polymerase chain reaction (PCR) method was used to amplify the SRY gene of single FNRBCs. Primer extension pre-amplification and nested PCR were used to amplify the SRY gene of the plasma DNA extracted from 65 samples of maternal blood. RESULTS: The detection rate of single FNRBCs was 90.20% (46/51). The concordance rates between real fetal sex and sex determined by amplification of the SRY gene from single cells and from free DNA analysis were 82.61% (38/46) and 90.77% (59/65), respectively. CONCLUSIONS: Single nucleated erythroblasts and free DNA in maternal blood are of fetal origin and can be valuable fetal material sources for non-invasive prenatal diagnosis.     

Isolation of epsilon-haemoglobin-chain positive fetal cells with micromanipulation for prenatal diagnosis

OBJECTIVES: The isolation and analysis of fetal cells in maternal blood during pregnancy is under investigation as a means of noninvasive prenatal diagnosis. The aim of our study was to detect fetal gender from maternal peripherial blood samples during pregnancy with the detection and analysis of epsilon-haemoglobin-chain positive fetal nucleated red blood cells (NRBCs) collected by a micromanipulator. Here we report our first results. DESIGN AND METHODS: We obtained maternal blood from 14 singleton pregnancies. After a double density gradient separation, magnetic activated cell sorting was performed by positive selection for nucleated red blood cells with anti-CD71. With the help of this enrichment step, followed by immunophenotyping with an anti-haemoglobin-epsilon monoclonal antibody, the isolation of the epsilon haemoglobin chain positive cells with micromanipulation could be done. We performed single cell fluorescent PCR analysis of these cells; we used primers for the amelogenin gene to detect fetal gender. We compared our findings with the results of amniocentesis. RESULTS: Fetal gender was successfully determined in 11 out of 14 cases; among them, in 2 cases with Klinefelter syndrome (47,XXY). CONCLUSION: The results of our study suggest that micromanipulation and QF-PCR analysis of anti-haemoglobin-epsilon fluorescent antibody stained fetal cells from maternal blood can be useful in prenatal diagnosis to detect fetal gender and promising to be improved to detect chromosomal abnormalities.     

母血中分离检测胎儿细胞的研究进展

进入母体血循环的胎儿细胞有4种.使用有效的方法从正常与异常妊娠母血中分离富集极少量的胎儿有核红细胞、滋养细胞、淋巴细胞和粒细胞.目前国外采用较多的有荧光激活细胞分选法、磁活化细胞分选法、免疫磁珠分离法、密度梯度离心法.分子生物学方法的引入.特别是聚合酶链反应及其衍生技术和荧光原位杂交技术提供了从母血中检测胎儿细胞敏感的方法并在无创伤性产前诊断方面得到一定的应用.     

孕妇外周血中胎儿有核红细胞的出现频率及其与孕周的关系

目的 探讨用检测孕妇外周血中的胎儿有核红细胞（ＮＲＢＣ）进行无创性产前诊断的最佳时间。方法 对４４名孕龄６～４０周的孕妇外周血进行不连续密度梯度离心,将分离后的细胞进行制片,显微镜下行有核红细胞计数,然后用显微操作法一一获取,进行Ｙ染色体特异性ＤＹＺ１基因的聚合酶链反应（ＰＣＲ）,以确定其胎儿来源。结果 ４４名孕妇中有１７例其外周血中检出有ＮＲＢＣ,分布于妊娠第９～２６周,其中以妊娠第１１～２０周     

双色共变性荧光原位杂交产前诊断胎儿唐氏综合征

目的 探讨双色共变性荧光原位杂交用于非侵入性产前诊断胎儿唐氏综合征的可行性。方法 对11例孕妇外周血中的胎儿有核红细胞进行抗血型糖蛋白磁珠直接标记,再经磁激活细胞分选法富集,以Y和21号染色体专一探针对分离的胎儿有核红细胞行双色共变性荧光原位杂交,预测胎儿21号染色体倍性和性别,并用羊水染色体核型分析结果,验证预测准确性。结果 11例胎儿21号染色体倍性均正常,与羊水染色体核型分析结果相符。其中5例为男性胎儿,男性胎儿有核红细胞数量为9－65个,平均为25个,男性胎儿有核红细胞纯度为1．4％－18．8％;6例为女性胎儿,孕妇外周血中未见男性胎儿有核红细胞;性别预测结果与羊水染色体型分析结果一致。结论 双色共变性荧光原位杂交用于分析胎儿21号染色体倍性及性别,诊断胎儿唐氏综合征准确、可靠。     

Development of noninvasive fetal DNA diagnosis from nucleated erythrocytes circulating in maternal blood

BACKGROUND: A considerable effort is being spent in developing noninvasive prenatal DNA diagnostic procedures. We recently reported that nucleated erythrocytes (NRBCs) can be enriched from maternal blood by a galactose-specific lectin method. In the present study, to prove that fetal NRBCs are definitely present in maternal blood and are a good source for fetal genetic diagnosis, we evaluated methods for lectin enrichment and subsequent fluorescence in situ hybridization (FISH) analysis through fetal gender determination. METHODS: Peripheral blood samples were collected from pregnant women (median 15, range: 10-18 weeks). From the blood samples, NRBCs were enriched based on galactose-specific lectin method. After detecting them by their morphology, NRBCs are separated and taken in a new glass slide by micromanipulator. We analyzed fetal gender using X and Y-chromosome-specific FISH probes. The results were compared with fetal gender analysis using Y-chromosomal sequences in maternal plasma. RESULTS: The fetal gender analyses by FISH in 20 pregnant women were all in accordance with the results from maternal plasma analyses. It is confirmed that fetal NRBCs were present in maternal blood and that 30.4% of NRBCs in maternal blood were fetal in origin. CONCLUSION: We have successfully carried out a noninvasive prenatal DNA diagnosis of fetal gender by using galactose-specific lectin method and subsequent FISH analysis.     

Detection of gamma-globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization

OBJECTIVES: Fetal nucleated red blood cells (NRBC) that enter the peripheral blood of the mother are suitable for non-invasive prenatal diagnosis. The application of peptide nucleic acid (PNA) probes for tyramide amplified flow fluorescence in situ hybridization (FISH) detection of gamma-globin mRNA in fixed fetal NRBC is investigated. METHODS: Hemin-induced K562 cells or nucleated blood cells (NBC) from male cord blood were mixed with NBC from non-pregnant women and analysed using both slide and flow FISH protocols. Post-chorionic villus sampling (CVS) blood samples from pregnant females carrying male fetuses were flow-sorted (2 x 10(6) NBC/sample). Y chromosome-specific PNA FISH was used to confirm that the identified gamma-globin mRNA stained cells were of fetal origin. RESULTS: Flow FISH isolated gamma-globin mRNA positive NBCs showing characteristic cytoplasmic staining were all Y positive. The amplification system generated a population of false positive cells that were, however, easy to distinguish from the NRBCs in the microscope. CONCLUSION: The gamma-globin mRNA specific PNA probes can be used for detection and isolation of fetal NRBCs from maternal blood. The method has additional potential for the study of gamma-globin mRNA levels or the frequency of adult NRBC (F cells) in patients with hemoglobinopathies.     

RhD血型的基因诊断及临床应用

目的 :建立RhD血型基因诊断的方法并对胎儿RhD血型进行产前诊断。方法 :采用PCR方法 ,对 4 89例供血者 (其中Rh-2 3例 ,Rh+ 4 6 6例 )外周血标本和 2 7例胎儿羊水 /脐血标本进行RhD基因外显子 10及外显子 7特异性片段的扩增。结果 :凡扩增出 136bp一条特异性片段者判断为Rh-;扩增出 136bp和 186bp两条特异性片段者判断为Rh+ 。 2 3例Rh-供血者有 2 2例扩增出 136bp特异性片段 ,4 6 6例Rh+ 供血者均同时扩增出 136bp和 186bp特异性片段 ,该方法的灵敏度为 10 0 % ,特异度为 95 6 5 % ,假阳性率为 4 .35 %。 2 7例胎儿羊水 /脐血标本的扩增结果 ,1例Rh-,2 6例Rh+ 。结论 :PCR方法检测RhD基因型简便、快速 ,且和血清学检测结果吻合率高 ,可用于胎儿RhD血型的产前诊断。